1. Field of the Invention
This invention relates generally to a method for a multiple fluorescent channel flow cytometer for subsequent analysis of samples, and in particular, to the use of microbeads with multiple fluorescent labels that serve as a reference for forward scatter and for fluorescence intensity for a plurality of fluorescent dyes.
2. Description of the Art
Flow cytometers are used to analyze biological cells and particles present in a fluid sample by intersecting a thin stream of the fluid by an illumination source, usually a laser beam. The resulting forward and right angle scattered and fluorescent light is analyzed with photomultiplier tubes (PMTs). The fluorescent channels of a flow cytometer, designated by Fl1, Fl2, Fl3 etc., are each set with barrier filters to detect a selected specific dye while filtering out signals from dyes that fluoresce at other wavelengths.
As discussed in the co-pending parent patent application, flow cytometers must be aligned and calibrated to obtain accurate, reproducible results. When more than one fluorescent dye is used, the instruments also require compensation for the fluorescence PMTs.
Alignment is the process of adjusting and focusing the optical and electrical components of the flow cytometer including the laser, lenses, mirrors barrier filters and PMTs, so that scatter and fluorescence signals have the lowest coefficient of variation of distribution (CV). Alignment of a flow cytometer can be determined by a dot plot of the forward and right angle scatter channels a shown by comparison of the dot plots of FIG. 1 (an unaligned instrument) and FIG. 2 (an aligned instrument).
Compensation is the process of electronically removing residual signals from fluorescent dyes in secondary fluorescence channels due to spectral overlaps no removed by barrier filters for the respective channels. Use of the flow cytometer's compensation circuits at an appropriate level changes the dot plot for the fluorescent channel Fl1 versus the fluorescent channel Fl2 from that shown in FIG. 3 (fluorescent microbeads (2) and (3) overlapping the boundaries designated by blank microbeads (1)) to that shown in FIG. 4 (the dot group is aligned with a blank or unlabeled sample in the secondary fluorescence channel). If the compensation is set too high, data from the sample may be lost (FIG. 5).
To perform accurate analysis with two or more fluorescent dyes, the electrical compensation circuit must be adjusted so that fluorescence emission which overlaps into other fluorescence channels may be subtracted from these other channels. For example, when fluorescein (FITC) and phycoerythrin (PE) are simultaneously employed as the fluorescent dyes, the green fluorescence channel for fluorescein may have a band pass emission filter of 520.+-.10 nanometers (nm) and the red fluorescence channel for phycoerythrin may have a band pass emission filter of 580.+-.10 nm. The emission spectrum of fluorescein is such that part of its fluorescence will be seen in the phycoerythrin fluorescence channel of 580.+-.10 nm, and to a lesser degree, part of the emission of phycoerythrin will appear in the fluorescein fluorescence channel of 520.+-.10 nm.
A particular problem associated with numerous samples measured by flow cytometry relates to naturally occurring fluorescence, i.e., autofluorescence, of the sample. For example, a wide variety of biological cells contain naturally occurring fluorescent compounds such as riboflavin. Such autofluorescence introduces an additional complexity to the flow cytometer compensation process, and tends to promote miscompensation (over-and/or under-compensation) in the respective fluorescence channels of the flow cytometer.
In co-pending application Ser. No. 07/374,435, the compensation circuits are adjusted such that the level of fluorescence in the fluorescence channels, other than the channel designated for particular fluorescent dye (the primary channel(s), is equal to the level of fluorescence of the sample prior to labeling the sample with fluorescent dyes. Autofluorescent microbeads, matching the fluorescence spectra and intensity of the unlabeled, naturally fluorescent sample to be measured, are run on the flow cytometer in the Fl1 versus Fl2 fluorescence channel dot plot or histogram display mode. The disclosure of the co-pending application and all patents and applications referred to herein are incorporated herein by reference.
Calibration of a flow cytometer with proper standards ensures that the results from samples will be comparable over time and between different instruments. For the calibration of the intensity of fluorescence signals to be independent of the specific instrument and instrument settings, the excitation and emission spectra of the calibration standards and of the samples being measured must be equivalent and the measurements on each must be made under the same instrument settings. In addition, as described in U.S. Pat. Nos. 4,714,682; 4,767,206; 4,774,189; 4,857,451; and copending U.S. applications Ser. Nos. 109,214 and 374,435, the disclosures of which hereby are incorporated by reference, when the calibration is made in terms of number of equivalent soluble fluorescent molecules, such correction factors, such as quenching and changes in extinction coefficient due to conjugation to other molecules, need not be taken into consideration.
Fluorescence calibration curves for flow cytometers may be constructed by plotting the mean or modal channels of the fluorescence intensity histograms of fluorescence microbead standards against the calibrated values of the number of equivalent soluble fluorescent dye molecules for the respective microbead standards, as shown in FIG. 6.
Although both small (0.1-2 microns) and large (2-50 microns) highly uniform microbeads are readily commercially available from a number of manufacturing companies, e.g., Seragen, Inc., Polysciences, Inc., and Interfacial Dynamics Corp. or have been described (U.S. Pat. Nos. 4,247,434 and 4,336,173), and the synthesis of fluorescent microbeads is taught in U.S. Pat. Nos. 4,157,323 and 4,179,685, fluorescent microbeads have not intended been used as uniform standards with multiple fluorescent labels on each microbead to adjust a flow cytometer for repeated use with a particular sample.
A kit of microbeads that match labeled cells is commercially available under the trademark CaliBrites.TM. from Becton Dickinson & Co. (Mountain View, Calif.), which consists of three microbead populations: (i) an unlabeled population, (ii) a fluorescein-labeled population, and (iii) a hycoerythrin-labeled population. None of these microbeads are calibrated in any way, and they are intended for standardization of a flow cytometer so that it provides repeatable, reproducible results with the same sample and dyes.
It is therefore a object of the present invention to provide a method for adjustment of a flow cytometer for analysis of selected samples, which may also comprise samples with multiple fluorescent labels, utilizing a single microbead population and enabling the flow cytometer to operate at high efficiency with respect to fluorescence data generated thereby, and in a manner achieving reproduceability of data which is independent of the specific instrument and time-frame of the data measurement and of the compensation of the instrument.
It is another object of the invention to provide a microbead reference standard and a kit having a single microbead population for carrying out such adjustment method.
Other objects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.